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Characterization of Natural and Affected Environments

Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale reclaimed water distribution systems Emily Garner, Jean McLain, Jolene Bowers, David Engelthaler, Marc A. Edwards, and Amy Pruden Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02818 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Environmental Science & Technology

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Microbial ecology and water chemistry impact regrowth of opportunistic pathogens in full-scale

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reclaimed water distribution systems

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Authors: Emily Garner1, Jean McLain2, Jolene Bowers3, David M. Engelthaler3, Marc A.

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Edwards1, Amy Pruden1*

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24061, United States

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2

Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia

Water Resources Research Center, University of Arizona, Tucson, Arizona 85719, United

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States

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3

Translational Genomics Research Institute, Flagstaff, Arizona 86005, United States

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*Corresponding Author (e-mail: [email protected])

1 ACS Paragon Plus Environment


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Abstract

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Need for global water security has spurred growing interest in wastewater reuse to offset demand

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for municipal water. While reclaimed (i.e., non-potable) microbial water quality regulations

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target fecal indicator bacteria, opportunistic pathogens (OPs), which are subject to regrowth in

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distribution systems and spread via aerosol inhalation and other non-ingestion routes, may be

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more relevant. This study compares the occurrences of five OP gene markers (Acanthamoeba

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spp., Legionella spp., Mycobacterium spp., Naegleria fowleri, Pseudomonas aeruginosa) in

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reclaimed versus potable water distribution systems and characterizes factors potentially

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contributing to their regrowth. Samples were collected over four sampling events at the point of

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compliance for water exiting treatment plants and at five points of use at four U.S. utilities

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bearing both reclaimed and potable water distribution systems. Reclaimed water systems

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harbored unique water chemistry (e.g., elevated nutrients), microbial community composition,

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and OP occurrence patterns compared to potable systems examined here and reported in the

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literature. Legionella spp. genes, Mycobacterium spp. genes, and total bacteria, represented by

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16S rRNA genes, were more abundant in reclaimed than potable water distribution system

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samples (p≤0.0001). This work suggests that further consideration should be given to managing

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reclaimed water distribution systems with respect to non-potable exposures to OPs.


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Table of Contents Art

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1 Introduction

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Growing need for sustainable water sources has spurred interest in direct and indirect

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potable reuse to supplement traditional surface and groundwater supplies. Approximately 1.6

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billion people globally live in watersheds impacted by water scarcity and, by 2050, it is projected

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that due to climate change and population increase, the number of people affected will roughly

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double.1 In these areas, wastewater reuse is particularly attractive to meet both potable and non-

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potable water demand. Non-potable reuse is already common in the U.S. for irrigation of

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agricultural and urban areas, groundwater recharge, and industrial reuse.2 While advanced

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treatments enable production of high-quality water, maintaining microbial water quality as water

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is transported to the point of use may present a greater challenge than that recognized for potable

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water and premise (i.e., building) plumbing, due to the unique qualities of reclaimed water,



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including high levels of growth-promoting nutrients, rapid decay of disinfectant residual,

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stagnation, and elevated distribution system retention times.3

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Where regulations exist, typically at the state level in the U.S., microbial water quality in

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reclaimed systems is typically characterized via monitoring of E. coli, Enterococci, or fecal or

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total coliforms.2 While these parameters track contamination from fecal bacteria, they are not

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good surrogates for opportunistic pathogens (OPs), which are non-fecal, such as Legionella

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pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, Acanthamoeba spp., and

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Naegleria fowleri.4 Although waterborne disease related to fecal pathogens has nearly been

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eradicated in most developed countries, OPs are now among the primary sources of tap water-

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related outbreak in the U.S. and elsewhere with developed water systems.5,6 OPs can infect

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humans via inhalation of aerosols or dermal, eye, or ear contact,7–10 which are more relevant than

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ingestion for non-potable reuse applications. L. pneumophila and M. avium are the causative

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agents of severe lung infections characterized by Legionnaires’ disease and M. avium complex,

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respectively.11,12 P. aeruginosa can infect hosts via the bloodstream, eyes, ears, skin, or lungs,8

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while Acanthamoeba spp. can cause infection of the eyes or central nervous system via

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inhalation or penetration of skin lesions.13 N. fowleri can infect the brain following entrance of

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water into the nasal cavity, with infections having been linked to nasal irrigation with neti pots

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and other hygienic or recreational activities where water can “get up the nose”.14 Exposure via

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aerosol inhalation could result from use of reclaimed water in cooling towers, spray irrigation,

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toilet flushing, fire suppression, and car washing.15–17 It has been demonstrated that the risk of

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Legionella infection associated with toilet flushing using reclaimed water exceeds a 10-4 annual

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risk, and for uses such as irrigation and cooling, large setback distances are necessary to achieve

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an annual risk of infection of 10-4.18 Further, dermal or eye and ear contact is feasible from use of


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reclaimed water for irrigation of athletic and recreational facilities, snowmaking, and toilet

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flushing.4,17 Presently, very little is known about the occurrence of OPs in reclaimed water

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distribution systems, with one field survey having documented their occurrence at the point-of-

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use,19 with the role of reclaimed water in transmitting OPs via non-ingestion routes representing

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an important knowledge gap.4,20

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While OPs are expected to be present at relatively low concentrations following treatment

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of recycled water, they are known to thrive in pipe biofilms and are generally tolerant of chlorine

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and other disinfectants, especially when residing in amoebae.21,22 OPs are also capable of growth

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under the extremely low organic carbon and nutrient concentrations characteristic of potable

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water.21 Stagnant conditions, which are common in reclaimed water systems due to seasonal

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shutdowns and intermittent demand, are also thought to trigger OP regrowth.23

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In addition to improved documentation of occurrence patterns of OPs in reclaimed

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distribution systems, fundamental understanding of how various physicochemical conditions

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relate to their regrowth potential during transport to the point of use is needed. The role of

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biostability (i.e., bioavailable nutrient content) of the water and other factors potentially

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stimulating regrowth of OPs in reclaimed water is of particular interest. Here we surveyed gene

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markers for Legionella spp., Mycobacterium spp., P. aeruginosa, Acanthamoeba spp., and N.

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fowleri in the distribution system point of entry (POE) and at five points of use (POU) at four

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U.S. utilities distributing reclaimed water for non-potable reuse and compared occurrences to

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corresponding municipal potable water systems over four sampling events. Quantitative

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polymerase chain reaction (qPCR) was employed to quantify specific OP gene markers of

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interest, while 16S rRNA amplicon sequencing and shotgun metagenomic sequencing provided

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broader context of microbial community structure and a means to explore other potential



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microbes of concern. The specific objectives were to 1) quantify regrowth in distribution systems

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by comparing OP gene copy numbers at the POE versus various POUs, 2) examine partitioning

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of OPs between bulk water and biofilms, 3) identify associations between water chemistry, water

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age and regrowth of OPs, and 4) characterize the relationship between the occurrence of OPs and

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the microbial community composition of the distribution system.

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2 Methods

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2.1 Site description, sample collection, and preservation

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Four U.S. utilities participated in this study (Table 1), with both the reclaimed and potable

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water distribution systems sampled in each city. Utilities were selected based on similar intended

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reclaimed water use (i.e., all utilities produced non-potable water primarily for irrigation

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purposes). For each potable or reclaimed system, samples were collected of freshly treated water

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at the point of compliance/POE to the distribution system and at five locations representing a

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range of water ages throughout the distribution system at the POU. Flushed bulk water samples

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were collected from POUs via distribution system sampling ports in sterile 1-L polypropylene

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containers prepared with 292 mg ethylenediaminetetraacetic acid (EDTA) and 48 mg sodium

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thiosulfate per liter sampled, to chelate metals and quench chlorine, which could kill cells,

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damage DNA, or otherwise inhibit or interfere with downstream molecular analyses. Samples for

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organic carbon analysis were collected in 250 mL amber glass bottles that were acid-washed and

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baked for five hours at 550ºC. Additional water was collected in separate acid-washed 250 mL

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bottles for other chemical analyses. For Utilities A and B, after collecting bulk water samples,

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biofilm samples were collected by, isolating and draining a section of accessible in situ pipe,

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inserting a sterile cotton-tipped applicator into the distribution system pipe (Fisher Scientific,

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Waltham, MA), pressing it firmly to the pipe’s surface, and in a single pass, swabbing the upper 6 ACS Paragon Plus Environment

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180º of the circumference of the pipe. The upper portion of the pipe was selected to avoid

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inadvertent collection of loose deposits accumulated at the bottom of the pipe. The swab was

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transferred directly to a sterile DNA extraction lysing tube and the stem snapped and severed to

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preserve only the sample end of the swab.

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Samples were shipped overnight on ice and processed within approximately 24 hours of

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sample collection. Samples for molecular analysis were concentrated onto 0.22 µm mixed

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cellulose esters membrane filters (Millipore, Billerica, MA). Filters were folded into quarters,

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torn into 1 cm2 pieces using sterile forceps, transferred to lysing tubes, and stored at -20ºC for

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later analysis. DNA was subsequently extracted using a FastDNA SPIN Kit (MP Biomedicals,

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Solon, OH). Biological activity reaction tests (BART; Hach, Loveland, CO) were used to

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examine the presence of active nitrifying, denitrifying, and sulfate-reducing bacteria.

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2.2 Water Chemistry

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Free chlorine, total chlorine, temperature, dissolved oxygen, pH, turbidity, and electrical

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conductivity were measured on-site using in-house resources used routinely by each participating

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utility. Upon receipt in the lab, 30 mL was subject to total organic carbon (TOC) analysis and 30

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mL was filtered through pre-rinsed 0.22 µm pore size mixed cellulose esters membrane filters

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(Millipore, Billerica, MA) for dissolved organic carbon (DOC) analysis. Biodegradable

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dissolved organic carbon (BDOC) was measured as previously described by Servais et al.24 but

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with the incubation time extended to 45 days. Samples were analyzed on a Sievers 5310C TOC

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analyzer (GE, Boulder, CO) according to Standard Method 5310C.25 Metals were measured

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using an Electron X-Series inductively coupled plasma mass spectrometer (ThermoFisher,

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Waltham, MA) according to Standard Method 3125B.25 Nitrate, nitrite, phosphate, and sulfate



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were quantified via a Dionex DX-500 ion chromatographer (Thermo Fisher, Waltham, MA)

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according to Standard Method 4110B.25

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2.3 Quantification of OPs

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OP gene copy numbers were quantified in triplicate reactions from DNA extracts using

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qPCR with published protocols for total bacterial and achaeal16S rRNA genes,26 Legionella spp.

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(23S rRNA),27 Mycobacterium spp. (16S rRNA),28 P. aeruginosa (ecfX and gyrB),29

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Acanthamoeba spp. (18S rRNA),30 and N. fowleri (internal transcribed spacer region).31 With the

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exception of N. fowleri, all protocols were validated for specificity in environmental matrices in

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a prior study.23 The specificity of the N. fowleri assay was confirmed by cloning and sequencing

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of qPCR products from a cross-section of positive samples (Table S1). In order to identify an

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optimized dilution for consistently minimizing the effect of PCR inhibition, a subset of DNA

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extracts (n=12) was initially analyzed at dilutions of 1:5, 1:10, 1:20, and 1:50, with a dilution

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factor of 1:10 found to yield optimum quantitation across extracts and qPCR assays. A triplicate

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negative control and triplicate standard curves of ten-fold serial diluted standards of each target

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gene ranging from 101 to 107 gene copies/µl were included on each 96-well plate. All qPCR

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negative controls failed to yield amplification above the limit of quantification for each assay.

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The limit of quantification was established as the lowest standard that amplified in triplicate in

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each run, and was equivalent to 10 gene copies per milliliter of bulk water and 103 gene copies

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per biofilm swab.

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2.4 16S rRNA gene amplicon sequencing

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Bacterial community compositions were profiled using gene amplicon sequencing with

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barcoded primers (515F/806R) targeting the V4 region of the 16S rRNA gene.32,33 Triplicate

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PCR products were composited and 240 ng of each composite was combined and purified using


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a QIAquick PCR Purification Kit (Qiagen, Valencia, CA). Sequencing was conducted at the

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Genomics Research Laboratory at the Biocomplexity Institute of Virginia Tech (BI; Blacksburg,

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VA) on an Illumina MiSeq using a 250-cycle paired-end protocol. Reads were processed using

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the QIIME pipeline34 and annotated against the Greengenes database35 (May 2013 release).

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Samples were rarefied to 10,000 randomly selected reads. Field, filtration, DNA extraction

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blanks, and a least one PCR blank per lane were included in the analysis.

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2.5 Shotgun metagenomic sequencing

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Shotgun metagenomic sequencing was attempted on the POE and greatest water age POU

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samples from each system on each collection date, however, all utilities’ potable samples, except

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Utility A, yielded insufficient DNA for analysis. Select potable samples were also sequenced.

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Libraries were prepared using Nextera XT (Illumina, San Diego, CA) and sequenced on an

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Illumina HiSeq 2500 using a 100-cycle paired-end protocol at BI. Samples were uploaded to the

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metagenomics RAST server (MG-RAST) and annotated against the RefSeq database using

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default parameters.36 Metagenomes are publicly accessible under the sample IDs listed in Table

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S2.

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2.6 Statistical Analyses

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Spearman’s rank sum correlation coefficients were calculated in JMP (SAS, Cary, NC) to

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assess correlations between OPs, water quality parameters, phyla, and corrosion bacteria using a

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significance cutoff of α=0.05. Given that this is a rank-based statistics test, all qPCR abundances

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below the limit of quantification were assigned a value of half of the limit of quantification. A

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Wilcoxon rank sum test for multiple comparisons was applied in JMP to determine differences

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between abundances of OPs across groups of samples. Unweighted UniFrac distances generated



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in QIIME were imported into PRIMER-E (version 6.1.13) for one-way analysis of similarities

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(ANOSIM) to determine taxonomic differences between groups of samples.

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3 Results and Discussion

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3.1 Overview of surveyed distribution systems

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The four reclaimed water distribution systems represented a range of U.S. geographic

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regions, climate zones, treatment schemes, and disinfectant types (Table 1). All utilities are

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located in climate zones that are warm seasonally or year-round and thus were candidates for

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potential regrowth of OPs, which generally prefer warmer water.21 Utility A used

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monochloramine as disinfectant residual, while all other utilities primarily used free chlorine. All

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potable water was derived from a combination of surface and groundwater sources. All utilities

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utilized advanced wastewater treatment to produce a relatively high quality finished product for

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distribution for the purposes of non-potable reuse.

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3.2 Physicochemical water characteristics

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The physicochemical water quality characteristics of distribution system samples (Table

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S3) suggested that, with the exception of Utility C, water in the reclaimed systems was warmer

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than the corresponding potable system, but only Utilities A and B were significantly warmer

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(p≤0.0321). TOC, DOC, and BDOC were consistently greater in reclaimed water than potable

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water (p≤0.0038). Average BDOC concentrations ranged from 2,137 to 6,094 ppb in reclaimed

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water and 15 to 1,522 ppb in potable water. The BDOC concentrations in reclaimed water were

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comparable to those reported in previous surveys of reclaimed water distribution systems, which

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ranged from 400 to 6,300 ppb BDOC.19,37

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Turbidity and conductivity were also elevated in reclaimed systems (p≤0.0002). Dissolved oxygen ranged from 5.5 to 7.7 mg/L on average in potable systems and 4.0 to 6.8 mg/L in 10 ACS Paragon Plus Environment

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reclaimed systems. Average total chlorine ranged from 0.7 to 3.5 mg/L in potable systems and

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0.3 to 2.7 mg/L in reclaimed systems. In reclaimed systems, where free chlorine was typically

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dosed for the purpose of serving as a secondary disinfectant residual, in reality it was susceptible

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to conversion to ambient chloramine residual because of reaction with elevated ammonia in the

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water. Total chlorine was significantly lower at POU sites than at the POE for all systems except

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Utility B (p≤0.0380), indicating decay of disinfectant residual. Distance from the POE to the

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POU, temperature, and TOC have all been previously identified as important factors contributing

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to enhanced decay of disinfectant residual in reclaimed systems.38

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3.3 Occurrence of OP Gene Markers

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The copy numbers of gene markers corresponding to five target OPs or genera containing

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multiple OP species that are commonly problematic in potable water distribution systems;39–42

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Legionella spp., Mycobacterium spp., P. aeruginosa, Acanthamoeba spp., and N. fowleri and

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total bacterial and archaeal 16S rRNA genes were determined via qPCR (Table 2). Given that

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qPCR provides an upper bound estimate of actual viable OPs, qPCR measurements are hereafter

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referred to in terms of abundance of their corresponding marker genes (i.e., gene copy numbers).

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Legionella spp., Mycobacterium spp., and 16S rRNA genes were more abundant in reclaimed

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than potable water distribution systems (p≤0.0001). In particular, Legionella spp. genes were

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widely detected in reclaimed water at the POU, ranging from 76-89% of samples from each

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utility being positive at an average of 3.4-4.4 log gene copies per milliliter. Legionella spp. genes

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were also widespread in Utility A’s potable water distribution system, with 80% of samples

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positive, though the average abundance was only 1.7 log gene copies per milliliter.

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Mycobacterium spp. genes were abundant in reclaimed water, with 59-79% of samples positive

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and average levels ranging from 2.5-3.7 log gene copies per milliliter. P. aeruginosa genes were



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more abundant in potable systems (p=0.0003), with up to 15% of samples positive from Utility

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B, but no more than 5% of samples positive from any reclaimed systems. N. fowleri genes were

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also notably widespread in Utility A’s potable (41% positive) and reclaimed (45% positive)

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distribution system samples, as well as Utility D’s potable samples (45% positive), though at

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relatively low abundances (2.1, 1.8, 1.3 log gene copies per milliliter on average, respectively).

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Although N. fowleri has been previously isolated from tap water, information is not available

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about the numbers of N. fowleri present in municipal water systems.43,44 It is notable that the

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frequency of detection of Legionella spp., Mycobacterium spp. and N. fowleri genes was

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generally highest in Utility A’s potable system, which was the sole utility employing

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monochloramine as the secondary disinfectant residual, whereas the others all utilized free

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chlorine. Maintaining a free chlorine residual of at least 0.2 mg/L has been proposed as a key

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strategy for control of N. fowleri.14 Disinfectant residual type may be an important factor

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influencing regrowth of these OPs.

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In a culture-based survey of four reclaimed distribution systems, Jjemba et al. found

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average log colony forming units per milliliter ranging from 0.6-1.9 for Legionella spp., 0.16-

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3.21 for Mycobacterium spp., and 0.001-0.009 for Pseudomonas spp.19 Though Jjemba et al. also

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found Legionella and Mycobacterium to be widespread in reclaimed water systems,

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concentrations were notably lower than those observed in the present study. However, it is to be

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expected that molecular tools provide an upper end estimate of pathogens, since they do not

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directly differentiate viable versus non-viable cells, while culture-based methods provide a lower

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end estimate, given that they do not capture viable but non-culturable (VBNC) cells. Legionella

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spp. commonly enter a VBNC state in water systems, which may relate to their characteristic

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oligotrophic status, given that VBNC is commonly induced by nutrient starvation.45 Previous


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studies have demonstrated that Legionella spp., Mycobacterium spp., and P. aeruginosa are all

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capable of entering a VBNC state, while culturable Legionella spp. CFU can be as much as two

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orders of magnitude less than corresponding viable cell estimates.46–48

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While there were no significant correlations among the different OPs in potable bulk

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water, Legionella spp. and Mycobacterium spp. genes were positively correlated with each other

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in reclaimed bulk water (ρ=0.4581, p

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